Molecular Cloning And Expression Of CDNA For Human Granulocyte Colony-Stimulating Factor (hG-CSF)

The human peripheral blood monocytes were stimulated by lipopolysaccharide (LPS) to produce hG -CSF mRNA and hG-CSF. The hG-CSF appeared in culture after 12 hours induction and to be maximum through 24 hours, The total RNA were isolated from human monocytes induced by LPS by using the method of acid guaninium— phenol— chloroform extraction and amplified specifically by RNA PCR technigue to obtain hG- CSF cDNA. The optimal annealing temperature detrained experimentally was 57° , at which the reaction product for hG-CSF cDNA was maximized and non-specific products were reduced. The G- CSF cDNA was inserted into the plasmid pUC18 and secretion expression vector, respectively, and thentransformed the recombinant plasmids into competent cells of an appropriate host strain. The recombinant plasmids were screened by gel electrophoresis and identified by Dot hybridization and southern blot analysis. After induction using IPTG, the hG- CSF protein was detected in the periplasmic space of the recombinant E. coli cells. These data provide evidene that hG-CSF cDNA has been cloned and expressed in E .coli successfully.